State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, PR China.
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, PR China; Jiangxi Institute for Food Control, Nanchang, 330001, PR China.
J Dairy Sci. 2020 Apr;103(4):3055-3065. doi: 10.3168/jds.2019-17590. Epub 2020 Feb 7.
Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 10 cfu/mL in pure culture and 4.5 × 10 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.
克罗诺杆菌属是婴儿配方粉中重要的机会致病菌，可引起新生儿和婴儿的许多严重疾病。本研究建立了一种基于双重信号放大策略的新方法，通过不对称尾式 PCR（AT-PCR）与滚环扩增（RCA）相结合，用于检测牛奶中的克罗诺杆菌属。通过 AT-PCR 产生的尾部单链 DNA 可启动 RCA，生成串联重复的 G-四链体序列。当存在可插入 G-四链体结构的荧光染料硫黄素 T 时，可通过微孔板读数器检测荧光信号。由于选择了用于 AT-PCR 的高度特异性引物，因此 AT-PCR 与 RCA 检测法具有特异性，可用于检测克罗诺杆菌属。在纯培养物中的检测限分别为 4.3×10 cfu/mL 和在添加牛奶中的检测限分别为 4.5×10 cfu/mL。发夹 DNA 中设计的固定序列使该 AT-PCR 与 RCA 检测法可通过修饰 PCR 引物的特异性，作为用于检测其他病原体的通用平台。
